Acetohydroxy acid synthase (AHAS) is an enzyme that catalyzes the initial step in the biosynthesis of isoleucine, leucine, and valine in bacteria, yeast, and plants. For example, the mature AHAS from Zea Mays is approximately a 599-amino acid protein that is localized in the chloroplast (see FIG. 1; SEQ ID NO:1). The enzyme utilizes thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD) as cofactors and pyruvate as a substrate to foam acetolateate. The enzyme also catalyzes the condensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate. AHAS is also known as acetolactate synthase or acetolactate pyruvate lyase (carboxylating), and is designated EC 4.1.3.18. The active enzyme is probably at least a homodimers. Ibdah et al. (Protein Science, 3:479-S, 1994), in an abstract, disclose one model for the active site of AHAS.
A variety of herbicides including imidazolinone compounds such as imazethapyr (PURSUIT®—American Cyanamid Company-Wayne, N.J.), sulfonylurea-based compounds such as sulfometuron methyl (OUST®—E. I. du Pont de Nemours and Company-Wilmington, Del.), triazolopyrimidine sulfonamides (Broadstrike™—Dow Elanco; see Gerwick, et al., Pestic. Sci. 29:357-364, 1990), sulfamoylureas (Rodaway et al., Mechanisms of Selectively of Ac 322,140 in Paddy Rice, Wheat and Barley, Proceedings of the Brighton Crop Protection Conference-Weeds, 1993), pyrimidyl-oxy-benzoic acids (STABLE®—Kumiai Chemical Industry Company, E. I. du Pont de Nemours and Company; see, The Pesticide Manual 10th Ed. pp. 888-889, Clive Tomlin, Ed., British Crop Protection Council, 49 Downing Street, Farmham, Surrey G49 7PH, UNITED KINGDOM), and sulfonylcarboxamides (Alvarado et al., U.S. Pat. No. 4,883,914) act by inhibiting AHAS enzymatic activity. (See, Chaleff et al., Science 224:1443, 1984; LaRossa et al., J. Biol. Chem. 259:8753, 1984; Ray, Plant Physiol. 75:827, 1984; Shaner et al., Plant Physiol. 76:545, 1984). These herbicides are highly effective and environmentally benign. Their use in agriculture, however, is limited by their lack of selectivity, since crops as well as undesirable weeds are sensitive to the phytotoxic effects of these herbicides.
Bedbrook et al., U.S. Pat. Nos. 5,013,659, 5,141,870, and 5,378,824, disclose several sulfonylurea resistant AHAS variants. However, these variants were either obtained by mutagenizing plants, seeds, or cells and selecting for herbicide-resistant mutants, or were derived from such mutants. This approach is unpredictable in that it relies (at least initially) on the random chance introduction of a relevant mutation, rather than a rational design approach based on a structural model of the target protein.
Thus, there is still a need in the art for methods and compositions that provide selective wide spectrum and/or specific herbicide resistance in cultivated crops. The present inventors have discovered that selective herbicide resistant variant forms of AHAS and plants containing the same can be prepared by structure-based modelling of AHAS against pyruvate oxidase (POX), identifying an herbicide binding pocket or pockets on the AHAS model, and designing specific mutations that alter the affinity of the herbicide for the binding pocket. These variants and plants are not inhibited or killed by one or more classes of herbicides and retain sufficient AHAS enzymatic activity to support crop growth.